Usher syndrome is the leading cause of hereditary deafness and of combined deafness and blindness in humans. The most severe form of the disease, Usher syndrome type 1 (USH1), has been linked to mutations in genes encoding myosin Vila, cadherin 23, protocadherin 15, sans and harmonin. These USH1 proteins are localized in hair cells, which are the specialized sensory cells of cochlea. A prevailing hypothesis is that USH1 proteins may participate in a macromolecular complex necessary for signal transduction and development of cochlear morphology. A key member of this complex is harmonin (USH1C), a protein containing PDZ domains, which are motifs mediating protein-protein interactions in a wide variety of molecules. Harmonin, via direct interactions with other USH1 proteins, may serve as a molecular scaffold for organizing critical signaling molecules in hair cells. Therefore, clues to understanding how genetic alterations in harmonin cause Usher's syndrome may be revealed in analyses of its protein-protein interactions. We have characterized a novel interaction between harmonin and Cav1.3 (L-type) Ca2+ channels, the primary voltage-gated Ca2+ channels in hair cells. Preliminary data indicate that harmonin directly interacts with Cav1.3, and that these two proteins may be colocalized in the apical membrane of cochlear hair cells. Given the importance of PDZ domain-containing proteins for targeting and clustering ion channels, the proposed research will test the hypothesis that the interaction between harmonin and Cav1.3 is essential for the proper targeting and localization of apical Cav1.3 channels in cochlear hair cells. That genetic disruption of the genes encoding Cav1.3 and harmonin independently lead to deafness in mice provides compelling rationale for the proposed research. The specific aims are to further characterize this interaction between Cav1.3 and harmonin utilizing biochemical and immunocytochemical techniques. A major goal is to develop a theoretical and experimental foundation on which to build an independent research plan focusing on the molecular and genetic alterations that can lead to cochlear dysfunction and hearing impairment. [unreadable] [unreadable] [unreadable]